Project Summary/Abstract: Despite the emergence of bacterial trehalose metabolism as a promising target for antibiotic and diagnostic development, there has been limited progress toward elucidating trehalose metabolic pathways, probing their roles in bacterial growth and virulence, and leveraging them for applications ranging from basic research to the detection and treatment of challenging infections. As established in the first period of this award, trehalose-based imaging probes and inhibitors can serve as powerful tools to drive progress in these areas. However, the challenges associated with the synthesis of trehalose analogues has severely impeded their development and applications. The long-term goal is to better understand bacterial trehalose metabolism and to exploit this knowledge for antibiotic and diagnostic development. The objectives of this project are to: (i) harness chemoenzymatic synthesis to create a functionally diverse toolbox of trehalose-based probes and inhibitors; and (ii) apply this toolbox to elucidate trehalose utilization characteristics of diverse bacteria and to develop inhibitors of trehalose metabolism in important pathogens. These objectives will be achieved through three specific aims: (Aim 1) Develop chemoenzymatic methods for the synthesis of trehalose and trehalose-6-phosphate (T6P) analogues; (Aim 2) Develop and apply detectable trehalose analogues to probe bacterial trehalose metabolism; and (Aim 3) Develop trehalose analogues as inhibitors of bacterial trehalose metabolism. In Aim 1, the TreT catalysis method for trehalose analogue synthesis developed in the prior award period will be improved in reaction scope and scale, and new chemoenzymatic methods will be developed to synthesize analogues of T6P, which is an essential metabolite in many bacteria. In Aim 2, labeled trehalose analogues will be chemoenzymatically synthesized and used to profile trehalose uptake characteristics in diverse bacteria and define the species-selectivity of trehalose-based imaging probes developed in the prior period. In Aim 3, based on results obtained in the first award period, rationally-designed trehalose-based inhibitors targeting trehalose utilization pathways in M. tuberculosis and C. difficile will be developed and evaluated using enzymatic and bacterial culture assays. This application is innovative because: (i) instead of traditional chemical synthesis, chemoenzymatic methods will be used to substantially increase the diversity, functionality, and accessibility of trehalose and T6P analogues for microbiology research; (ii) while trehalose analogues have been applied nearly exclusively to mycobacteria, this project will expand the usage of these tools to other types of bacteria. This research is significant because it will produce new synthetic methods, tool compounds, and strategies to investigate and target trehalose metabolism, which is critical to growth and virulence in numerous bacterial pathogens. This project will continue to support the interdisciplinary training of CMU and USM undergraduate students in a biomedical research area of high importance, and it will enhance the research environment at CMU, in part through continued development and utilization of a biosafety level 3 (BSL-3) tuberculosis research facility.